Gel Electrophoresis and Transfer Combination using Conductive Polymers and Method of Use

ABSTRACT

A precast gel and blotting membrane combination unit and method of use. The device includes two plates, each plate having a conductive opaque region with conductive polymers and a transparent region having static-dissipative polymers. Between the plates are a gel matrix and blotting membrane. The device is placed in a tank capable of both performing the electrophoresis phase and transfer phase of a western blot. During the electrophoresis phase, current flows from a pair of electrophoresis electrodes to separate proteins by size. The user can visualize the extent of protein separation by observing a tracking dye through the transparent region. After the electrophoresis phase, voltage is switched to a pair of transfer phase electrodes. The device allows current to flow through the conductive opaque regions of the plates to transfer separated proteins to a blotting membrane directly after electrophoresis without having to remove or reorient the device in the tank.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of, and claims priority to, U.S. patentapplication Ser. No. 15/186,110, entitled “Gel Electrophoresis andTransfer Combination Using Conductive Polymers and Method of Use,” filedJun. 17, 2016, the contents of which are incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to gel electrophoresis andtransfer with a precast gel and blotting membrane combination unit usingconductive polymers.

BACKGROUND OF THE INVENTION

Although western blotting is a common technique, there are still manyissues that arise from the protein transfer step. These difficultiesinclude the introduction of air bubbles when placing gels on membranes,and the gels often tear when they are transferred from a precast orother casted gel setting to a separate protein transfer membrane,especially in the case of thinner gels that are employed to reduce theamount of protein needed. These complications can be devastating whenthe availability of the protein sample is limited, no more protein isavailable, or when the protein is a clinical specimen. The source ofmany of the problems with the prior art can be traced to the need toremove the gel from the gel cassette in order to accomplish the transferstep. It has not been possible to eliminate this step in the prior artbecause the gel cartridges are made of materials that arenon-conductive/insulative and are used to support the gels.Additionally, researchers often prefer to watch the electrophoretic stepprogress, which has limited the motivation to invent electrophoretic gelsystems incorporating non-transparent conductive materials.

It would be desirable to have a one-step separation and transfer westernblot apparatus and method of separating and transferring proteins thatutilize a single combination of a precast gel and protein transfermembrane. Hence, it would be advantageous to have a means to avoid theintroduction of air bubbles, and to avoid the tearing of gels duringtransfer.

There have been many attempts to simplify the separation and transfer ofproteins for analysis using various types of apparati and techniques.U.S. Pat. No. 4,994,166 to Fernwood et al. describes a single apparatusfor slab gel electrophoresis and blotting, both of which are performedin a single tank cell, which contains separation electrodes alongopposing vertical walls, and blotting electrodes arranged horizontallyabove and below the level of gel placement. The cell is operated inseparatory and blotting modes, in which separatory and blottingelectrodes are separately energized. Fernwood requires porous gelsupports to allow the electric field to pass through the membrane. Inaddition, the top plate transfer electrode must be removed from contactwith the buffer solution during the protein separatory phase.

U.S. Pat. No. 5,102,524 to Dutertre describes a multiple electrophoresismethod, where different sets of electrodes are used in a two-stepprocess to first separate biomolecules and then to transfer them to adeposition membrane.

U.S. Pat. No. 5,593,561 to Cognard describes a multiple electrophoresismethod for controlled migration of biomolecules and transfer thereof toa membrane in a vessel, containing a plurality of parallel elongatedelectrodes. The first electric field, established between electrodes,provides means for macromolecular separation in a gel, and the secondelectric field, perpendicular to the first, provides means fortransferring the biomolecules onto the membranes. In the describedmethod, electrodes and transfer membranes are first assembled in thevessel, which is then filled with gel. After the separation ofbiomolecules in the gel, and the proteins are transferred to themembrane, the gel is liquefied, dissolved, or decomposed, which allowsfor the removal of the membrane. Cognard's invention is for use withoutprefabricated gel and membrane combination units.

U.S. Pat. No. 8,173,002 to Margalit describes a dry blotting system totransfer proteins onto a transfer membrane. The system does not includean electrophoresis device, so the device does not allow the user tovisualize the separation and transfer phases in a single device. Thedevice requires the user to transfer the gel to a transfer membrane onthe blotting device. Margalit teaches the use of electrically conductingpolymers, but not in combination with a single device that bothseparates proteins and transfers the proteins to a transfer membrane.

U.S. Patent Appl. Pub. No. 2006/0042951 to Ohse discloses an apparatusto separate and transfer proteins via the use of a fine groove, atransferring electrode, and a transparent conductive material having athickness of approximately 0.1 μm. The apparatus includes a pair ofseparating electrodes for causing a substance in a sample to move alonga passage, and a pair of transferring electrodes for causing thesubstance in the sample to be transferred to the capturing material byelectrophoresis. The conductive material is not capable of serving asthe support structure due to its thickness of approximately 0.1 μm,which would not have sufficient strength to serve effectively as thesupporting walls for a gel. The separation and blotting is performed inan electrophoresis buffer and does not make use of a gel slab or gelslab assembly, which are commonly used for western blots.

U.S. Pat. No. 6,602,391 to Serikov discloses an apparatus and method forcapillary separation of biomolecules and post-separation blotting.However, Serikov does not disclose the use of a slab gel where the usercan view the separation of biomolecules and transfer the macromoleculeto a blotting membrane for western blotting.

Conductive polymers have previously been described, but not inconjunction with electrophoresis and blotting. Ates et al. Describesvarious applications of conducting polymers in “Conducting Polymers andtheir Applications” (Current Physical Chemistry, 2012, 2, 224-240).International Pat. Appl. No. PCT/EP2013/065163 to Jung discloses aconductive polymer composition and transparent electrode for anantistatic layer. International Pat. Appl. No. PCT/KR2008/002236 to Kimdiscloses a conductive polymer for use as a transparent electrode and amethod of fabricating the electrode using an ink jet spray method. U.S.patent application Ser. No. 13/616,804 to Kim et al. discloses atransparent panel and method of manufacturing a transparent panel wherea conductive polymer layer is formed to make a transparent electrode.U.S. patent application Ser. No. 15/017,540 to Woodham discloses anapparatus and method for using a gel transfer combination havingtransparent conductive polymers and to separate proteins during anelectrophoresis phase and thereafter transfer proteins to a transfermembrane after electrophoresis without transferring the gel from anelectrophoresis apparatus to a separate protein blotting transferapparatus. Opaque conductive polymers also have previously beendescribed, but not in conjunction with electrophoresis and blotting. Forexample, U.S. Pat. No. 5,609,315 to Lepore describes electricallyconductive opaque sheets made of polyimide and U.S. Pat. No. 4,702,371to Hoshi describes electrically conductive portions of a plasticmaterial to prevent electrostatic breakdown of electrical componentssuch as those in integrated circuits.

One challenge in creating a single combination gel electrophoresis andprotein transfer unit involves creating an apparatus where the user canvisualize the degree of protein separation during electrophoresis andthereafter transfer the proteins to a blotting membrane withoutphysically transferring the gel to the membrane. For such an apparatusto perform both electrophoresis and protein transfer, the plates mustform the structural support for the gel, and also be able to transfercurrent through the gel supporting plates to a blotting membrane. Thechallenge is that the electrical current required to transfer proteinsto the blotting membrane must run perpendicular to the current requiredto separate proteins during electrophoresis. Gel supporting plates thatcan be made rigid, transparent, and conductive would be ideal for use insuch a gel and membrane combination unit.

One promising material that can provide rigidity, transparency, andconductivity are transparent metal compositions such as indium tin oxide(ITO). Compositions like ITO have been used in some applications whereboth conductivity and transparency are required, however, a considerablecompromise must be made between conductivity and transparency. Likewise,transparent polymers such as conductive transparent plastics could beused, but there is also a compromise between conductivity andtransparency. Furthermore, transparent conductive polymers are costlyand may be cost prohibitive for commercial application such for the usein western blots.

Another problem with separating proteins within an electrophoresis gelis that if the user does not carefully monitor the protein separationphase, proteins may run off of the gel into the electrophoresis buffer.One way to prevent proteins from running off the gel is to have theelectrophoresis power source on a timer so that after a pre-set amountof time has elapsed, the current shuts off. However, due to severalvariables (such as gel thickness and temperature), a timed shut-off maynot allow the proteins to be optimally separated if the timer is set tooshort. If the timer is set too long, the proteins may run off the gelinto the buffer. In addition, once the electrophoresis timer stops,proteins begin to diffuse within the gel. If the protein transfer stepis not performed immediately after electrophoresis, protein bands maynot be sufficiently defined.

Given the disadvantages of using a timer to end the electrophoresisphase, an alternative is to use an optical sensor that is capable ofdetecting the dye front of a sample loading buffer with known separationcharacteristics that is loaded into the wells of the gel as part of theprotein running sample. When the optical sensor detects the dye front,the sensor communicates this information to the power source and shutsthe current off, or controls the current in some other manner. Differenttypes of electrophoresis power control devices have previously beendescribed. One optical sensor linked power control device is describedin PCT Appl. No. PCT/US2013/030220 to Asare-Okai et al. Asare-Okaidiscloses a controller with a sensor that is positioned adjacent to agel matrix. The sensor includes a light source for emitting light intothe gel matrix and a light detector disposed adjacent to the lightsources for detecting light from the illuminated gel matrix. Thecontroller is connected to a power source that provides a current acrossthe electrophoresis phase electrodes. The controller is operable forturning off electrical power to the power source based on a change inthe light emitted from the gel matrix due to migration of the trackingdye through the illuminated gel matrix.

U.S. Pat. No. 5,120,419 to Papp discloses a photoelectricelectrophoresis controller. The controller is triggered by moleculardyes that are sensed by the photodetector when the dye reaches apredetermined position in the gel matrix, characterized by an observingphotocell spaced from a reference photocell for comparison.

U.S. Pat. No. 5,268,568 to Lee discloses a marker dye band detector forgel electrophoresis using balanced light emitters. The device is capableof detecting a marker dye used in gel electrophoresis when the markerdye has reached a specific position in the gel. The device activates analarm or shuts off the power source when the sensor detects the dye.Aare-Okai, Papp, and Lee do not disclose the use of optical sensors in acombination apparatus that can both separate and transfer proteins to ablotting membrane.

In view of the above limitations in the field, there currently exists aneed in the industry for a device and associated method that can performgel electrophoresis and protein transfer to a blotting membrane in oneprecast gel and transfer membrane combination unit.

All patents, patent applications, and non-patent references disclosed inthe background and description of this application are herebyincorporated by reference for all purposes in their entireties.

SUMMARY OF THE INVENTION

The present invention advantageously fills the aforementioneddeficiencies by providing gel electrophoresis and protein transfer usinga single precast gel and blotting membrane combination unit usingconductive polymers, the use of which provides a fast, reliable, andeasy method to perform a hands-free protein separation followed by anefficient transfer of proteins to a blotting membrane.

The technology is defined as any technology, invention, know-how,method, composition, device, machine, product, consumable, formula andany combination thereof that relates to any use of conductive,semiconductive, and/or dissipative materials in the structure of adevice for supporting an electrophoretic gel in which a blottingmembrane is positioned adjacent to the gel, thereby permittingelectrical current to flow through the gel in one direction during theprotein separation phase and after the protein separation phase hascompleted, the current flows through conductive plate regions in adirection perpendicular to the direction of the flow of the currentduring the separation phase. This is accomplished without removing thegel from the precast gel and membrane combination unit.

The apparatus includes a first gel matrix supporting plate and a secondgel matrix supporting plate substantially parallel to the first plate.The two plates have at least one region that is made from an opaqueconductive polymer. The two plates sandwich a gel matrix and a blottingmembrane, where the gel matrix is capable of separating proteins by sizewithin the gel matrix when an electric current flows between electrodeson opposite sides of the y-axis of the gel matrix. The blotting membraneis capable of immobilizing proteins transferred from the gel matrixafter protein separation without physically transferring the gel matrixto a blotting membrane after protein separation.

In one embodiment, the gel matrix supporting plate further includes atransparent static-dissipative polymer region. The transparentstatic-dissipative region is adjacent to the opaque conductive regionalong the y-axis of the opaque conductive polymer region. In thisembodiment, the user can visualize a pre-stained molecular marker ladderhaving a loading dye that results in a dye front that migrates down thegel along the y-axis during electrophoresis.

One advantage of having both a transparent region (which isstatic-dissipative) and an opaque conductive region in a single unit isthat these two features in combination allow the user to (1) see themolecular ladder separation through the transparent region while (2) notbe constrained by the compromise usually encountered by having to choosebetween thickness, transparency, and conductivity in the region of theplate required for protein transfer during the transfer phase. Bycombining plates that have both a transparent region and an opaqueconductive region, the user can determine the extent of proteinseparation by visualizing the extent of pre-stained molecular ladderseparation (and migration of the dyes within the molecular ladderloading sample), while also having an apparatus where current can flowthrough opaque conductive regions to transfer the separated proteins tothe blotting membrane. Since this molecular ladder does not need to betransferred to the blotting membrane for later protein analysis as theuser can choose to run an alternate molecular marker ladder of choice inanother well for transfer to the membrane, the transparentstatic-dissipative region of the plate does not have to be conductive.Therefore, only a narrow transparent region is required for the user toobserve the pre-stained proteins and dye front, which serves as a proxyfor the adjacent protein separation occurring behind the opaqueconductive region.

In yet another embodiment, the apparatus also includes a thin layer of aless conductive gel (i.e. high percentage polyacrylamide) between theelectrophoresis gel matrix and the blotting membrane. Theelectrophoresis gel matrix may include gels made from polyacrylamide,bis-Tris, Tris-acetate, etc. Immunoblotting membranes include those madefrom nitrocellulose, polyvinylidene difluoride (PVDF), etc.

Another embodiment is for a system to perform a single stepelectrophoresis and transfer of proteins. The system uses plasticinsulators, a buffer tank and buffer tank lid. The tank includes anelectrode assembly having a pair of electrophoresis separation phaseelectrodes, a pair of transfer phase electrodes, positive and negativeelectrode chambers, anode and cathode buffers, a cooling chamber, and aprogrammable power source to switch voltage from the separation phaseelectrodes to the transfer phase electrodes.

In yet another embodiment, the device includes a loading dye sensor tosense a dye front along the electrophoresis gel matrix. The sensor isconnected in communication with circuitry in a detector that iselectrically connected to the power source. When the sensor detects adye front within the electrophoresis gel, the detector triggers thepower source to switch from applying voltage from across the first andsecond separation phase electrodes to across the first and secondtransfer phase electrodes. This feature is advantageous because the usercan program the power source first to separate proteins in the gelmatrix, and then transfer the separated proteins to the blottingmembrane. The sensor detects when the dye front has progressed apredetermined distance, which allows the user to avoid reliance on atimer, or avoid reliance on manually switching the voltage from theseparation phase electrodes to the transfer phase electrodes.

In yet another embodiment, there is a method for protein separation andpost-separation protein transfer onto a membrane. The method includesproviding the gel and membrane combination unit described above within aliquid receptacle tank in a first orientation. After the user places theunit in the tank, the user applies a voltage across a pair of separationphase electrodes to separate biomolecules along a gel matrix, where thevoltage across the pair of separation phase electrodes causes current toflow along the y-axis of the gel matrix in the first orientation as theconductivity of the gel is higher than that of the conductive polymersthat encase it. The voltage causes the separating of biomolecules withinthe gel matrix by size. The voltage is then discontinued across the pairof separation electrodes. A voltage is then applied across a pair oftransfer phase electrodes to transfer proteins from the gel matrix to ablotting membrane. This causes current to flow along a z-direction ofthe gel matrix in the first orientation. The current transfersbiomolecules from the gel matrix to a blotting membrane. The steps ofseparating the biomolecules and transferring the biomolecules to theblotting membrane are performed without removing the unit from the tank,thereby combining the steps of electrophoresis and transfer in a singleliquid receptacle tank without having to reorient the device between theseparating step and transferring step.

The present invention owes its uniqueness to the fact that the apparatusand methods employ conductive polymers to house a precast gel membranecombination unit that acts as an insulator in one scenario, and anelectrode in another scenario. This is advantageous for a device thatseparates proteins via electrophoresis in one direction using separationphase electrodes and transfers proteins perpendicularly out of the gelmatrix to a blotting membrane via applying a voltage across a pair oftransfer phase electrodes. The invention uses innovative conductivepolymers with particular resistivity and along with transparentstatic-dissipative polymers in western blotting applications to solvefundamental problems found in current devices and methods. The presentinvention permits convenient, one-step protein separation and transferof proteins for later analysis on a blotting membrane.

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, which are intended to be read inconjunction with both this summary, the detailed description, and anypreferred embodiments specifically discussed or otherwise disclosed.This invention may however be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided by way of illustration only toconvey the full scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention willbecome appreciated as the same becomes better understood with referenceto the specification, claims, and drawings herein:

FIG. 1 shows a side view of the general setup of a precast gel andblotting membrane combination unit for electrophoresis and transfer,which includes a high conductivity gel and filter paper;

FIG. 2 shows a side view of a general setup of a precast gel andblotting membrane combination unit for electrophoresis and proteintransfer showing directional electric current flow (arrows) to separateproteins within the gel matrix during electrophoresis;

FIG. 3 shows a side view of a general setup of a precast gel andblotting membrane combination unit for electrophoresis and proteintransfer showing directional electric current flow during proteintransfer to a blotting membrane perpendicular to the electric currentflow that occurred during the electrophoresis phase;

FIG. 4A shows a front view of the general setup of the precast gel andblotting membrane combination unit for electrophoresis and proteinseparation having transparent and non-transparent regions of a gelmatrix support plate;

FIG. 4B shows a front view of the general setup of the precast gel andblotting membrane combination unit for electrophoresis and proteinseparation having only a non-transparent plate;

FIG. 5 shows a cross sectional side view of the precast gel and blottingmembrane combination unit within an electrophoresis and transfer tank;

FIG. 6 shows a perspective view of the electrophoresis and transfer tankwithout the precast gel and blotting membrane combination unit placedinside the tank;

FIG. 7 shows a side view of an embodiment of the precast gel andblotting membrane combination unit having a conductive wire mesh;

FIG. 8 shows a perspective view of an embodiment of the precast gel andblotting membrane combination unit having a conductive wire mesh;

FIG. 9 shows a top view of an embodiment of the precast gel and blottingmembrane combination unit having projections on one plate to create agap for the gel and blotting membrane.

DETAILED DESCRIPTION OF THE DRAWINGS

The invention now will be described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may however be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

It will be understood that, although the terms first, second, third etc.may be used herein to describe various elements, components, regions,layers, and/or sections, these elements, components, regions, layers,and/or sections should not be limited by these terms. These terms areonly used to distinguish one element, component, region, layer, and/orsection from another element, component, region, layer, and/or section.

It will be understood that the elements, components, regions, layers andsections depicted in the figures are not necessarily drawn to scale.

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting of the invention.As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” or “includes” and/or “including” when used in thisspecification, specify the presence of stated features, regions,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof.

Furthermore, relative terms, such as “lower” or “bottom,” “upper” or“top,” “left” or “right,” “above” or “below,” may be used herein todescribe one element's relationship to another element as illustrated inthe Figures. It will be understood that relative terms are intended toencompass different orientations of the device in addition to theorientation depicted in the Figures.

Unless otherwise defined, all terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. It will be further understood that terms, such asthose defined in commonly used dictionaries, should be interpreted ashaving a meaning that is consistent with their meaning in the context ofthe relevant art and the present disclosure, and will not be interpretedin an idealized or overly formal sense unless expressly so definedherein.

Exemplary embodiments of the present invention are described herein withreference to idealized embodiments of the present invention. As such,variations from the shapes of the illustrations as a result, forexample, of manufacturing techniques and/or tolerances, are to beexpected. Thus, embodiments of the present invention should not beconstrued as limited to the particular shapes of regions illustratedherein but are to include deviations in shapes that result, for example,from manufacturing. The invention illustratively disclosed hereinsuitably may be practiced in the absence of any elements that are notspecifically disclosed herein.

The present invention is directed to electrophoretic separation andtransfer of proteins or other biomolecules with a precast gel andblotting membrane combination unit 10 using conductive polymers in atleast one region of the unit. The polymers for use in the gelelectrophoresis and transfer apparatus include conductive andtransparent static-dissipative plastics. Electrophoresis may beperformed using a variety of methods, including well-known western blottechniques employing the use of sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE).

FIG. 1 shows a side view of one embodiment of the general setup of theprecast gel matrix and blotting membrane combination unit 10 forseparation of proteins during an electrophoresis phase and transfer ofproteins during a transfer phase. The combination unit 10 has a firstgel matrix supporting plate 2 and a second gel matrix supporting plate4. Each plate 2, 4, has two distinct regions, (1) an opaque conductiveregion 86, 88, and (2) a transparent static-dissipative region 90, 92.The gel matrix 6 and blotting membrane 12 are sandwiched between the twoplates 2, 4. Additionally, the gel 6 and membrane 12 may be separated bya thin layer of a low conductive (i.e. high percentage polyacrylamide)gel 8. During the electrophoresis protein separation phase, currentflows from the upper surface 20 of the gel 6 to the lower surface 18 ofthe gel 6 (the current represented by the vertical arrows pointingdownward in FIG. 2). This is accomplished by the particularconductivity/resistivity of the conductive polymers, in which theirresistivity is higher than the gel 6. After the protein separationphase, the voltage is switched so that current flows through theconductive opaque regions 86, 88, thereby transferring the separatedproteins to the blotting membrane 12 (the current represented by thehorizontal arrows pointing right FIG. 3).

The Conductive Opaque and Transparent Static-Dissipative Regions

Generally, the polymers used to create support structures, such as thefront and rear support plate of electrophoresis gels, are plastics, andtherefore electrically insulating. However, there is a special class ofpolymers that intrinsically conduct or dissipate electricity at levelsmuch higher than semiconductors (up to 1000 S/cm), and theirconductivities/resistivities can be controlled through different methodsof production. Conductive or dissipative polymers are organic polymersthat conduct or dissipate electricity. Specifically, they offerelectrical conductivity/dissipativity less than metals, and can haveproperties of plastics, such as transparency. The electrical properties(i.e. resistivity) can be fine-tuned using organic synthesis methods anddispersion techniques. Types of organic conductive polymers includepolyacetylene, poly(pyrrole)s (PPY), polyanilines, poly(thiophene)s(PT), poly(3,4-ethylenedioxythiophene) (PEDOT), poly(p-phenylenesulfide) (PPS), poly(acetylene)s (PAC), poly(p-phenylene vinylene)(PPV), and others, such as those described in U.S. Pat. App. Pub. No.20110050623 to Lee et al., and U.S. Pat. App. Pub. No. 20090032107.Generally, the electrical conductivity of a polymer is created byremoving or adding an electron from the polymer's conjugated π-orbitalvia doping and the delocalization of electrons along the polymerbackbone.

If a voltage is applied across typical insulating polymers, the polymermay discharge in a dangerous manner. To prevent discharge, thetransparent polymer region of the plate where voltage is applied shouldbe made from static-dissipative polymers, which generally haveresistivity between 10⁶ to 10⁹ ohms per square. Static-dissipativepolymers have little or no initial charges, prevent discharge to andfrom human contact, and are thus safer to use thannon-static-dissipating polymers. In the present invention, by using atransparent static-dissipative polymer for the transparent regions, theuser can visualize ladder migration in an electrophoresis gel and yetstill remain safe when voltages are applied during the electrophoresisand transfer phases. Examples of static-dissipative polymers aredisclosed in U.S. Pat. No. 4,729,925, U.S. Pat. No. 4,762,941, U.S. Pat.No. 5,968,656, U.S. Pat. No. 5,828,931, U.S. Pat. No. 5,798,060 U.S.Pat. No. 7,214,757, and U.S. Pat. No. 8,653,177, the disclosures ofwhich are incorporated by reference herein. Compositions ofstatic-dissipative polymers comprise or consist of at least one ofpolyamide-imides, polytetrafluoroethylene (PTFE), polyetheretherketones,polyetherimides, polyetherimides and their derivatives.

Gel Matrix

The electrophoresis gel matrix 6 is a slab gel and can be made from avariety of electrophoresis-supporting materials, including acrylamide,bisacrylamide, polyacrylamide, Tris-glycine, bis-Tris, Tris-acetate,cellulose acetate, agarose, silica, and other materials, as well as suchmaterials in treated or derivatized form known among those skilled inthe art. Agarose gels would typically be used for DNA and RNA analysis,and polyacrylamide, Tris-glycine, bis-Tris, and Tris-acetate gels forprotein analysis. Typical resolving gels for protein analysis are madefrom between 6% and 15% polyacrylamide. The same techniques that havebeen described for use of the invention for protein separation andblotting can also be used for separation and blotting of DNA and RNAwith the use of the appropriate gel.

In preferred embodiments, a bis-Tris polyacrylamide gel is comprised of10% to 12% bis-Tris polyacrylamide and a Tris-acetate polyacrylamide gelis comprised of approximately 7%-10% Tris-acetate polyacrylamide, butvalues may lie outside these ranges depending on the size of the proteinthat the user wishes to analyze or probe in the sample. The dimensionsof the electrophoresis gel 6 are typically rectangular and in apreferred embodiment are approximately 10 cm×10 cm, but may varydepending on the number of samples to be run simultaneously, the type ofsample, and the sample volume.

In a preferred embodiment, the electrophoresis gel and transfer membranecombination unit 10 is less than 0.5 cm thick, but could also bedesigned thicker. Additionally, a stacking gel (not depicted) on top ofthe electrophoresis gel 6 may also be included above the primaryelectrophoresis gel 6, and made from a lower percentage of theequivalent corresponding material used in the electrophoresis gel 6.Stacking gels are well known in the art as a tool to concentrate andpack all the proteins of the sample into one band before separationoccurs. Differences in the pH and acrylamide concentration at theinterface of the stacking and separating gel 6 functions to compress thesample at the interface and provides better resolution and sharper bandsin the separating gel 6. A standard electrophoresis plastic comb isplaced in the primary electrophoresis gel or stacking gel to createwells 84 in the gel where the user loads the protein-containing samples.

Blotting Membrane

Adjacent to the gel 6 is a blotting membrane 12, also known as atransfer membrane, immobilization membrane, or blotting paper. Theblotting membrane 12, may be made from a variety of blotting materials,such as nitrocellulose, PVDF, nylon, or other materials known in the artused for immobilizing proteins on a sheet.

As depicted in FIG. 1, the gel 6 and membrane 12 may be separated by athin low conductivity gel 8. The low conductivity gel 8 is positionedbetween the blotting membrane 12 and electrophoresis gel 6. The lowconductivity gel 8 prevents the direct contact of the blotting membrane12 with the electrophoresis gel 6 during the electrophoresis phase.Since proteins have a high affinity to blotting membranes 12, the lowconductivity gel 8 prevents proteins from binding to the surface of themembrane 12 during electrophoresis.

Gel Supporting Plates

Each plate 2, 4 is made from a combination of opaque conductivematerials and static-dissipative materials. Opaque conductive materialscomprise or consist of at least one of conductive polyethylenes such asTivar® 1000 EC and Lennite® CN, conductiveacrylonitrile-butadiene-styrenes (ABS) such as Absylux® CN, conductiveacetal (polyoxymethylene or POM) copolymers such as Pomalux® CN-F orPomalux® CN-SS, conductive polypropylenes such as Propylux® CN-P orPropylux® CN-F, conductive polyetherimides such as Tempalux® CN,conductive polyaryletherketones such as TecaPEEK™ ELS, conductivepolycarbonates such as Zelux® CN-P or Zelux® CN-SS, (all currentlyavailable through Boedeker Plastics, Inc.) or, other known conductivepolymers, all of which can can be manufactured with volume resistivitiesin the range of 10³ to 10⁵ ohm-cm. The generic equivalents of the abovementioned conductive polymers are also suitable for use in theconductive plates 2, 4. In addition, the plates 2, 4, may be doped withconductive powder, such as carbon or stainless steel powder, or may bemade from intrinsically conductive polymers.

Transparent static-dissipative compositions include compositions madefrom polyanilines, polypryrrols, polythiophenes, polycarbonates such asPC-300™ available through SciCron Technologies, or other knowntransparent static-dissipative polymers. Each plate 2, 4 has an opaqueregion 86, 88 made from the opaque conductive polymers. In a preferredembodiment, the polymer used for the opaque conductive region 86, 88 hasa volume resistivity in the range of approximately 10³-10⁵ ohm-cm,whereas the transparent dissipative region 90, 92 has a volumeresistivity in the range of approximately 10⁵-10⁹ ohm-cm. Since theconductivity of the opaque conductive regions 86, 88 that form thesupport plates of the gel 6 is less than that of typical conductors(such as metals) the opaque conductive region 86, 88 may be considered asemi-conductor (at least compared with the conductivity of typicalmetals that have a volume resistivity typically around 10⁻⁶ ohm-cm andthe volume resistivity of insulators, typically around 10¹⁸ ohm-cm).This semi-conductive quality of the opaque conductive region 86, 88,where the opaque conductive region 86, 88 is not as conductive as thegel 6, cause the plates 2, 4 to act as insulators during theelectrophoresis step and do not interfere with the protein separationelectrophoresis phase. During the protein separation electrophoresisphase, the electrical current takes the path of least resistance throughthe gel 6 and not through the plates 2, 4 because the plates 2, 4 havehigher resistivity than the gel 6. This feature of the opaque conductiveregion being less conductive than the electrophoresis gel overcomesproblems addressed in the prior art, such as in U.S. Pat. No. 4,994,166to Fernwood, where plate electrodes made from highly conductivematerials (such as those made from metals) may nullify an electric fieldaround a gel, and therefore could prevent separation of proteins duringelectrophoresis.

Surrounding the opaque conductive region 86, 88 is the transparentstatic-dissipative region 90, 92. The opaque conductive region 86, 88 ofeach plate 2, 4 is positioned adjacent to the transparent region 90, 92.The opaque conducive region 86, 88 depicted in FIG. 4, shows thetransparent static-dissipative region framing the opaque conductiveregion 86, 88 on three sides: (1) above the opaque region 86, 88 alongthe x-axis of the gel, (2) on one side of the opaque conductive region86 along the y-axis of the gel, and (3) below the opaque region 86, 88the along the x-axis of the gel. The plates 2, 4 may have pedestals 80(See FIG. 9) on their side edges to allow for the creation of a cavitythat forms the boundaries of the electrophoresis gel 6 and blottingmembrane 12.

The transparent static-dissipative regions 90, 92 allows the user toobserve the loaded samples in the gel 6 and also observe the migrationof a molecular marker ladder 94 in a lane adjacent to the opaqueconductive region 86, 88 so that the user can deduce the extent ofprotein separation during electrophoresis. Moreover, as typical plasticsare insulative, they cannot be used adjacent to the conductive materialsas charge could build up during the protein transfer step, therebycreating a discharge hazard. The combination of both transparentstatic-dissipative polymers with opaque conductive polymers reduces thishazard. In addition, the entirety or near entirety of the plates 2, 4could be made from transparent conductive or semi-conductive polymers ora combination of the two, or a combination of transparent and opaquetransparent conductive and semi-conductive polymers.

In some embodiments, such as the one illustrated in FIG. 4A, only asingle loading lane 110 is visible in the transparent static-dissipativeregion 90, 92. A single loading lane 110 is all that should be necessaryto visually determine the extent of protein separation that has occurredduring electrophoresis because protein separation is correlated withpre-stained molecular marker ladder separation.

To ensure a substantially equal electric field emanating from all areasof the opaque conductive regions 86, 88 during the transfer phase, theopaque conductive region 86, 88 may have one or more thin wires (ornanowires) 76, 82 disposed on the plates' outer surfaces, as depicted inFIG. 8.

In one embodiment of the combination unit 10, adjacent to the blottingmembrane 12 is a filter paper 60. The filter paper 60 is sandwichedbetween the blotting membrane 12 and the second gel matrix supportingplate 4. Filter paper 60, when wet, acts as an ion reservoir, therebyaiding in the transfer of biomolecules to the blotting membrane 12during the transfer phase. Filter paper 60 also ensures that theblotting membrane 12 stays wet. The blotting membrane 12 and filterpaper 60 may be pre-wet prior to assembly of the combination unit 10,often using a solution containing methanol or other wetting buffer. Thefilter paper 60 may also be wet from the buffer solution used during theelectrophoresis and transfer phases.

Returning again, FIG. 4A, FIG. 4B depicts the front view of the precastgel and blotting membrane combination unit 10 for electrophoresis andtransfer. During the electrophoresis protein separation phase, asvoltage is applied across the top and bottom of the gel 6, proteinsmigrate vertically from the top of the gel 6 to the bottom of the gel 6along its y-axis. In one embodiment, there are two non-conductiveinsulative plastic strips 26, 28 that flank the sides of the gel 6, orcould be made from projections or pedestals of the plates themselves.The strips 26, 28 are also sandwiched between the plates 2, 4, or areextensions of the plates to establish the space for and direct currentthrough the gel 6 during the electrophoresis phase. These strips,projections, or pedestals also provide structural support and rigidityto the unit 10. The transfer of proteins during the transfer step isalong the z-axis, (i.e. perpendicular to the y-axis direction of proteinseparation and also perpendicular to the x-axis loading well directionof the gel).

The gel and blotting membrane unit 10 is placed in a tank apparatus 30in order to allow current to flow along the y-axis and z-axis of the geland blotting membrane unit 10. One such apparatus to perform bothelectrophoresis and blotting is illustrated in FIG. 5. FIG. 5 depictsthe tank apparatus 30 together with the precast gel and blottingmembrane combination unit 10, which together is a system for performingboth the electrophoresis separation phase and transfer phase. The tankapparatus 30 is a liquid receptacle that includes a front panel 32, rearpanel 68, first side panel 70, second side panel 72, bottom panel 66,lip 58 on the rear panel 68, and a lid (not shown). The lip 58 may be avariety of shapes but in a preferred embodiment is substantiallyU-shaped along the inner walls of the first and second side panels 70,72 of the tank apparatus 30.

Typical for a protein separation gel, the combination unit may besubmerged in an electrolyte containing buffered solution such asTris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE),2-(N-morpholino)ethanesulfonic acid (MES), or3-(N-morpholino)propanesulfonic acid (MOPS). Other buffers may be useddepending on the type of gel used in the precast gel and blottingmembrane combination unit 10. For example, a TAE buffer may be used forTris-acetate polyacrylamide gels, whereas MES or MOPS buffers may beused for bis-Tris polyacrylamide gels. The buffers in this system shouldbe efficient for both the electrophoresis phase and transfer phase.

The tank apparatus 30 has an upper chamber 34 and a lower chamber 36. Afirst separation phase negative electrode (cathode) 38 is disposedwithin the upper chamber 34. A second separation phase positiveelectrode (anode) 40 is disposed within the lower chamber 36. The firstand second separation phase electrodes 38, 40 are each connected to aprogrammable power source 108 to power the separation electrodes 38, 40.The desired voltage for electrophoresis between the first and secondseparation phase electrodes 38, 40 is generally between 80 and 150volts, but may be lower or higher depending on the desired rate ofseparation. The power source 108 employs switching means for electricalisolation of the separation phase electrodes 38, 40 from the transferphase electrodes 50, 52. The switching means include manual switchingmeans through the use of a button, toggle or any otherelectro-mechanical means to change current from one set of electrodes toa different set of electrodes. The power source may also includeautomatic means to switch the applied voltage from one set of electrodesto a different set of electrodes, such as by automatic switching after apre-set amount of time, or switching after an optical detector sensesthe presence of a dye front in the gel. The power source is connected tooptical detector and to the anodes 38, 50 and cathodes 40, 52, of theapparatus.

As depicted in FIGS. 4A and 4B, and with respect to the inclusion of anoptical sensor as a means for automatic voltage switching, the gel andblotting membrane combination unit 10 may include an optical sensor 100,which can be connected to a separate optical detector 104 havingcircuitry to detect dye within the gel 6. The optical detector 104 maybe physically connected to the sensor 100 by wire 102 or connectedwirelessly. The optical detector 104 is connected to a power source 108.The optical detector may also be connected to the power source 108 bywire 106 or wirelessly, and communicate power control information to thepower source 108, such as through WiFi, radio signal, a physicalconnection, or any other of a number of known means in the art tocommunicate information to control output.

The use of optical sensors and detectors in electrophoresis systems isknown in the art and may be incorporated into the present invention. PCTApplication No. PCT/US2013/030220, U.S. Pat. No. 5,120,419, and U.S.Pat. No. 5,268,568 all disclose different devices and methods to use anoptical sensor in an electrophoresis device, the disclosures of whichare incorporated by reference herein. In the present embodiment, as thesensor 100 and detector 104 detect the presence of a dye front 98, thedetector transmits a signal to the power source 108 to switch voltagefrom the electrophoresis electrodes 38, 40 to the transfer electrodes50, 52.

In another embodiment, such as the one depicted in FIG. 4B, the gel andblotting membrane combination unit 10 does not include a transparentregion in the plate 2, 4, but instead only includes the opaqueconductive region 86, 88. In this type of embodiment, the user would notvisualize the molecular ladder 94 and therefore not visually confirmseparation of proteins. However, one or more sensors 100 are placedwithin the conductive opaque regions 86, 88 to detect the presence of adye. The optical detector 104 triggers the power source 108 to switchvoltage from the separation phase electrodes 38, 40 to the transferphase electrodes 50, 52, when the detector 104 detects the dye. As dyepasses in front of a sensor 100 positioned at a strategic location alongthe gel 6, the presence of dye indicates sufficient protein separationand the next step of protein transfer to a membrane can begin.

To accomplish electrophoresis and transfer of proteins within the tankapparatus 30, the upper chamber 34 and lower chamber 36 are each filledwith a buffer solution 56. The chambers 34, 36 are electricallyconnected to each other via the electrically conducting gel and transfercombination unit 10. The buffer solution 56 and gel 6 allow negativecharges to pass from the first separation electrode 38, through thebuffer 56 in the upper chamber 34, then through gel 6 to buffer 56 inthe lower chamber 36, and finally to the second separation phaseelectrode 40. This flow of current, which generally forces to migratedown through the gel, is accomplished in part due to the relatively lowconductivity of the conductive and static-dissipative polymers housingthe gel, which have a higher resistance than the gel 6.

In one embodiment, the buffers 56 will have a volume resistivity ofapproximately 10-200 ohm-cm and gels may have similar volumeresistivities, typically between 100-300 ohm-cm. The opaque conductiveregions 86, 88 will have volume resistivities in the range of 10³ to 10⁵ohm-cm, and the static-dissipative regions 90, 92 will have volumeresistivity of 10⁵ to 10⁹ ohm-cm. These ranges will allow for theelectric current to flow through the gel during the separation phasewhen a power source is applied to separation electrodes 38, 40 ratherthan through the first and second plates 2, 4. Then, when voltage isapplied across the transfer phase electrodes 50 and 52, the electriccurrent flows substantially perpendicular to the length of the y-axis ofgel 6 through the membrane 12, to the second plate 4, thereby allowingthe proteins to adhere to the blotting membrane 12 during the transferphase.

While these ranges have been described in terms of exemplaryembodiments, it is to be understood that they are not limiting, whereasany embodiment in which the buffer 56 and gel 6 have a reasonably lowerresistivity than the conductive polymers (i.e. plates 2, 4) that housethem, and conversely that the conductive polymers (i.e. plates 2, 4)have a reasonably higher conductivity than the buffer 56 and gel 6, willallow for the described separation and transfer phases.

The rear panel 68 has one or more openings 74 in its lower region toallow the buffer solution 56 from the lower chamber 36 to fill up to thelower surface 18 of the gel 6 to provide an electrical connection fromthe second separation electrode 40 to the gel 6. The buffer solution 56in the upper chamber 34 and lower chamber 36 may be the same buffersolution, or may be different buffer solutions, where in someembodiments, the buffer solution 56 in the upper chamber 34 may includean antioxidant.

The wires electrify the buffer solution 56, thereby causing the solutionin the upper chamber 34 to act as the cathode (−) and solution in thelower chamber 36 to act as the anode (+). Proteins in a sample buffercontaining sodium dodecyl sulfate (SDS), or other buffers that are wellknown in the art, impart proteins with negative net charge so that whenthe proteins are in the gel 6, the proteins move from the cathode (−) 38to the anode (+) 40 due to the electromotive force (EMF) created by thepower source. By placing proteins in wells 84 and applying an electricfield along the y-axis of the gel, the proteins move through the gel 6at different rates, determined largely by their masses.

As illustrated in FIG. 5, the buffer solution 56 in the upper chamber 34and lower chamber 36 are not in buffer contact with each other, but arestill in electrical contact. The buffer solutions 56 in the top andbottom chambers 34, 36 are physically separated by the gel and blottingmembrane combination unit 10. Gaskets 44, 46 prevent the buffer solution56 from filling the entirety of the tank apparatus 30. A rear panelgasket 46 is disposed on the inner surface of the rear panel 68 of thetank apparatus 10. The rear panel gasket 46 prevents buffer solution 56from contacting a transfer electrode 52, which would cause unwantedelectrical current flow during the separation phase if buffer filled theentire tank and all sides of the combination unit 10. Additionally, theapparatus 30 may include a lip gasket 44 disposed along the outersurface of the lip 58. The lip gasket 44 prevents buffer solution 56,necessary for the separation phase, from contacting the cooling solution54 used in the cooling chamber 42. The cooling chamber can be filledwith water, buffer, or other types of coolant. Gaskets in the preferredembodiments may be made from rubber, silicone, and other flexiblematerials commonly used to form seals that prevent liquid seepage.

The rear panel gasket 46 is positioned so that when the precast gel andmembrane combination unit 10 is placed within the tank apparatus 30, theouter surface of the second plate 4 is pressed against the gasket 46.The lip gasket 44 is positioned so that the inner surface of the firstplate 2 is pressed against the lip gasket 44. As shown in FIG. 6, therear panel gasket 46 is a continuous loop along the inner surface of therear panel 68. The lip gasket 44 has an open shape with a bottom regionconnected to two side regions. The lack of a rubber sealing structure onthe top allows the buffer 56 to be in electrical contact with the top ofgel 6. In sum, the gaskets 44, 46 are positioned such that when theprecast gel and membrane combination unit 10 is placed correctly withinthe tank apparatus 30, the gaskets 44, 46 form seals that keep the upperchamber 34 and lower chambers 36 (necessary for the electrophoresisphase) separate from the cooling chamber 42 and other structuresrequired during the protein transfer phase.

Another feature of the embodiment that prevents the upper and lowerchambers 34, 36, from being in electrical contact with the coolingchamber 42 is that the first plate plate 2 is larger than the secondplate 4 as depicted in FIGS. 1, 5, and 7-9. In a preferred embodiment,the first plate is approximately 12 cm×12 cm and the second plate 4 isapproximately 10 cm×10 cm (approximately the same dimensions of the gel6). The larger first plate 2 allows for the first plate 2 to contact thelip gasket 44, and allows the smaller second plate 4 to be in contactwith the rear panel gasket 46. The smaller second plate 4 allows thebuffer solution 56 to pass over and under the second conductive plate 4in the upper chamber 34 and lower chamber 36, respectively, but not passthrough the larger first plate 2. This setup prevents the buffersolution 56 from entering into the cooling chamber 42 and contacting thetransfer electrodes 50, 52 used during the protein transfer phase.

After the protein separation phase, when the proteins have beenseparated by size vertically along the y-axis of gel 6, voltage isswitched from the separation electrodes 38, 40 to transfer electrodes50, 52, which forces the proteins to transfer from the gel 6 to theblotting membrane 12. The opaque conductive region 86 is in electricalcontact with a first transfer electrode 50 connected to the power source108, and the second conductive opaque region 88 is in electrical contactwith a second transfer electrode 52.

In the embodiment shown in FIG. 5, the first transfer electrode 50 is anarc shaped metal brace to provide sufficient tension to hold the precastgel and membrane combination unit 10 in place against the gaskets 44, 46to form the different chambers 34, 36, 42 of the apparatus 30. Thetransfer electrode 50 could also be a separate element from thestructure that braces the precast gel and membrane combination unit 10inside the tank 30 against the various gaskets 44, 46. In otherembodiments, the first transfer electrode 50 may be separated into twoor more brackets (e.g. one on the left side, one on the right side) sothat the user can still view the gel 6, but the precast gel andcombination unit 10 is still held firmly in place within the tank 30.Other types of braces can also be placed inside the cooling chamber 42without departing from the spirit of the invention as long as there istension and electrical contact between the transfer electrode 50 and thefirst plate 2.

The second transfer electrode 52 is disposed along the inner surface ofthe rear panel 68 and acts as the anode (+) during the protein transferphase. In the embodiment shown in FIG. 5, the second transfer electrode52 has a spring or recoil action so that the transfer electrode 52 makessufficient contact with the second plate 4. In other embodiments, thetransfer electrode 52 may be a separate element from an element havingthe spring or recoil action to help brace the precast gel and membranecombination unit 10 inside the tank 30 against an opposing bracingmember.

An electrical power source 108 connects the first transfer electrode 50with the second transfer electrode 52 and voltage is applied across thetransfer electrodes 50, 52 such that the first plate 2 acts as thecathode (−) and second plate 4 acts as the anode (+) during the proteintransfer phase. The power source 108 shall provide enough current toachieve sufficient transfer of the proteins from the gel 6 to thetransfer membrane 12 housed within the precast gel and membranecombination unit 10. Typical voltages applied across the transferelectrodes 50, 52 to achieve sufficient current flow to transferproteins to the blotting membrane 12 are between 5 to 30 volts, buthigher voltages can also be applied without damaging the gel 6 orblotting membrane 12.

FIG. 6 is a perspective view of the tank apparatus 30 without theprecast gel and blotting membrane combination unit 10. The combinationunit 10 would be placed adjacent to (on the left side) of the lip 58having the lip gasket 44. Since the first plate 2 is larger than thesecond plate 4, the first plate 2 lays on the outer surface, while theelectrophoresis gel 6, low conductivity gel 8, transfer membrane 12 andfilter paper 60 are positioned within the inner cavity of the lip 58 andthe second plate 4 is pressed against the rear panel gasket 46.

FIGS. 7 and 8 show another embodiment of a precast gel and membranecombination unit 10. Within or on the opaque conductive region 86 are aplurality of conductive wires or conductive mesh 76, which may bearranged in a grid or array. The mesh 76 distributes electric currentalong the opaque conductive region 86. In addition to a wire meshdisposed on or within the first opaque conductive region 86 is a secondwire mesh 82 that may be disposed on or within the second opaqueconductive region 88 to evenly distribute charge along the second opaqueconductive region 88. The wire meshes 76, 82 ensure that electric chargeis evenly distributed along the conductive regions 86, 88.

FIG. 9 illustrates another example of the precast gel and membranecombination unit 10. This embodiment includes two pedestal projections80 in the static-dissipative region of the first plate 2 abutting theinner surface of the second plate 4. The second plate 4 rests over thepedestals 80, thereby forming a cavity or gap between the inner surfaceof the first plate 2 and the inner surface of the second plate 4. Thegap size may vary depending on the desired thickness of the gel 6. In apreferred embodiment, the gap between the first and second plates 2, 4ranges from about 0.1 cm to about 0.5 cm. The gap holds the variouspreviously described components needed for proper separation andblotting of proteins in the precast gel and membrane combination unit10, such as the electrophoresis gel 6, low conductivity gel 8, transfermembrane 12, and filter paper 60.

While the invention has been described in terms of exemplaryembodiments, it is to be understood that the words which have been usedherein are words of description and not of limitation. As is understoodby persons of ordinary skill in the art, a variety of modifications canbe made without departing from the scope of the invention defined by thefollowing claims, which should be given their fullest, fair scope.

I claim:
 1. A method for separation and post-separation transfer ofbiomolecules to a blotting membrane, the method comprising the steps of:providing a device within a liquid receptacle tank in a firstorientation, wherein the device comprises: a first gel matrix supportingplate having at least one region made from an opaque semi-conductivepolymer; a second gel matrix supporting plate substantially parallel tothe first gel matrix supporting plate, the second gel matrix supportingplate having at least one region made from an opaque semi-conductivepolymer; a gel matrix between the first and second gel matrix supportingplates, wherein the gel matrix is capable of separating biomoleculessuch as proteins by size within the gel matrix when an electric currentflows between electrodes of opposite polarity on opposing sides of thegel matrix; and, a blotting membrane between the gel matrix and at leastone of the first and second gel matrix supporting plate, wherein theblotting membrane is capable of immobilizing biomolecules such asproteins transferred from the gel matrix after separation withoutphysically transferring the gel matrix to the blotting membrane;applying a voltage across a pair of separation phase electrodes toseparate biomolecules along a gel matrix, wherein the voltage across thepair of separation phase electrodes causes current to flow along ay-axis of the gel matrix in the first orientation and separatebiomolecules within the gel matrix according to size; separatingbiomolecules within the gel matrix by size; discontinuing applying thevoltage across the pair of separation electrodes; applying a voltageacross a pair of transfer phase electrodes to transfer biomolecules fromthe gel matrix to a blotting membrane, wherein applying the voltageacross the pair of transfer phase electrodes causes current to flowalong a z-direction of the gel matrix in the first orientation;transferring biomolecules from the gel matrix to a blotting membrane;wherein the steps of separating the biomolecules and transferring thebiomolecules to the blotting membrane is performed without removing thedevice from the liquid receptacle tank, thereby combining the steps ofelectrophoresis and transfer in a single liquid receptacle tank withouthaving to reorient the device between the separating step andtransferring step.
 2. The method of claim 1 wherein the steps ofdiscontinuing applying the voltage across the pair of separationelectrodes and applying a voltage across a pair of transfer phaseelectrodes is triggered by detecting the presence of dye within the gelmatrix.